Sustained p16INK4a expression is required to prevent IR-induced tumorigenesis in mice.

Exposure of murine and human tissues to ionizing radiation (IR) induces the expression of p16INK4a, a tumor suppressor gene and senescence/aging biomarker. Increased p16INK4a expression is often delayed several weeks post exposure to IR. In this context, it remains unclear if it occurs to suppress aberrant cellular growth of potentially transformed cells or is simply a result of IR-induced loss of tissue homeostasis. To address this question, we used a conditional p16INK4a null mouse model and determined the impact of p16INK4a inactivation long-term post exposure to IR. We found that, in vitro, bone marrow stromal cells exposed to IR enter DNA replication following p16INK4a inactivation. However, these cells did not resume growth; instead, they mostly underwent cell cycle arrest in G2. Similarly, delayed inactivation of p16INK4a in mice several weeks post exposure to IR resulted in increased BrdU incorporation and cancer incidence. In fact, we found that the onset of tumorigenesis was similar whether p16INK4a was inactivated before or after exposure to IR. Overall, our results suggest that IR-induced p16INK4a dependent growth arrest is reversible in mice and that sustained p16INK4a expression is necessary to protect against tumorigenesis.

Human cytidine deaminase as an ex vivo drug selectable marker in gene-modified primary bone marrow stromal cells.

Naturally occurring drug resistance genes of human origin can be exploited for selection of genetically engineered cells co-expressing a desired therapeutic transgene. Their non-immunogenicity in clinical applications would be a major asset. Human cytidine deaminase (hCD) is a chemoresistance gene that inactivates cytotoxic cytosine nucleoside analogs, such as cytosine arabinoside (Ara-C). The aim of this study was to establish if the hCD gene can serve as an ex vivo dominant selectable marker in engineered bone marrow stromal cells (MSCs). A bicistronic retrovector comprising the hCD cDNA and the green fluorescent protein (GFP) reporter gene was generated and used for transduction of A549 cells and primary murine MSCs. Analysis of transduced cells demonstrated stable integration of proviral DNA, more than 1000-fold increase in CD enzyme activity, and drug resistance to cytosine nucleoside analogs. In a mixture of transduced and untransduced MSCs, the percentage of retrovector-expressing cells could be increased to virtual purity (>99.5%) through in vitro drug selection with 1 microM Ara-C. Increased selective pressure with 2.5 microM Ara-C allowed for enrichment of a mixed population of MSCs expressing approximately six-fold higher levels of GFP and of CD activity when compared with unmanipulated engineered MSCs. Moreover, engraftment and endothelial differentiation of these in vitro selected and enriched gene-modified marrow stromal cells was demonstrated by Matrigel assay in vivo. In conclusion, these findings outline the potential of human CD as an ex vivo selection and enrichment marker of genetically engineered MSCs for transgenic cell therapy applications.

Selection of drug-resistant transduced cells with cytosine nucleoside analogs using the human cytidine deaminase gene.

Hematopoietic toxicity produced by most anticancer drugs limits their potential for curative therapy. We have shown previously that the human cytidine deaminase (CD) gene can confer drug resistance in murine bone marrow cells (BMCs) to the nucleoside analog, cytosine arabinoside (ARA-C). In the present study, as the first objective we showed that the CD gene can also render drug resistance in BMCs to related analogs, 2',2'-difluorodeoxycytidine (dFdC) and 5-azadeoxycytidine (5-AZA-CdR). As a second objective, we investigated the potential of ex vivo selection with cytosine nucleoside analogs of CD-transduced BMC. The goal of this approach was to enrich the fraction of CD-transduced BMCs so as to increase the transgene expression and level of drug resistance before transplantation. This strategy may have the potential to circumvent the problem in clinical gene therapy of low level of gene transfer and adequate long-term gene expression. Using a bicistronic retroviral vector containing the CD and the green fluorescent protein (CDiGFP), we transduced murine L1210 leukemic cells. All three analogs, ARA-C, dFdC, and 5-AZA-CdR were demonstrated in vitro to enrich (>95%) the population of leukemic cells expressing the GFP transgene. However, with CD-transduced primary murine BMCs cultivated at high cell density we observed that in vitro selection with ARA-C was not possible due to release of CD into the culture medium at amounts that were sufficient to inactivate the analog. The CD-containing medium produced a chemoprotective effect on mock BMCs as shown by lack of significant growth inhibition in the presence of ARA-C. However, at low cell density in a cell mixture containing CD-transduced cells, the mock BMCs showed marked drug sensitivity to ARA-C as determined by clonogenic assay. Selection with ARA-C was shown to significantly increase the CD enzyme activity in transduced BMC. These results suggest that CD gene has the potential to be a good selectable marker and a possible tool for chemoprotection in cancer gene therapy.

Potential of ribozymes against deoxycytidine kinase to confer drug resistance to cytosine nucleoside analogs.

Hematopoietic toxicity is the dose-limiting side effect produced in cancer chemotherapy with deoxycytidine nucleoside analogs. Deletion of the deoxycytidine kinase (dCK), results in a drug resistance phenotype to these analogs. An interesting gene therapy strategy to confer drug resistance to cytosine nucleoside analogs would be to specifically inactivate the dCK in normal hematopoietic stem cell. In this study, we designed hammerhead ribozymes that can specifically cut and downregulate the murine dCK mRNA. Three different ribozymes were identified and shown to cleave in vitro the dCK RNA. After introduction of ribozyme cDNA into murine L1210 leukemic cells by retroviral transfer, two of the ribozymes showed some capacity in reducing dCK activity. However, analysis of transduced L1210 clones showed that the significant reduction in the dCK mRNA was not sufficient to confer drug resistance to cytosine arabinoside. Nevertheless, these results provide a new avenue of modulating the dCK enzyme activity and with improved modifications may have the potential for use in gene therapy to confer drug resistance to deoxycytidine analogs.

Coexpression of rat glutathione S-transferase A3 and human cytidine deaminase by a bicistronic retroviral vector confers in vitro resistance to nitrogen mustards and cytosine arabinoside in murine fibroblasts.

The transfer of drug resistance genes into hematopoietic cells is an experimental approach to protect patients from drug-induced myelosuppression. Because anti-cancer drugs are often administered in combination to increase their clinical efficacy, vectors that express two drug resistance genes are being developed to broaden the spectrum of chemoprotection. We have constructed a bicistronic vector, MFG/GST-IRES-CD (MFG/GIC) coexpressing rat glutathione S-transferase (GST) A3 isoform (rGST Yc1) and human cytidine deaminase (CD). Murine NIH 3T3 fibroblast cells transduced with this vector were evaluated for their resistance to nitrogen mustards and cytosine nucleoside analogs. GIC-transduced polyclonal cell populations (GIC cells) demonstrated marked increases in selenium-independent glutathione peroxidase (peroxidase) and CD activities, as well as increased resistance to melphalan (2.3-fold), chlorambucil (3.4-fold), and cytosine arabinoside (Ara-C) (8.1-fold). After selection with Ara-C, the peroxidase and CD activities of GIC cells were augmented 2.6- and 2.9-fold, respectively, in comparison with unselected cells, and the resistance to melphalan, chlorambucil, and Ara-C was further increased to 3.7-, 5.9-, and 53-fold, respectively. Melphalan selection of GIC cells likewise augmented their peroxidase (2.3-fold) and CD (1.9-fold) activities. GIC cells proliferated in the simultaneous presence of melphalan and Ara-C at drug concentrations that completely inhibited the growth of untransduced cells. The growth rate of unselected GIC cells exposed to the drug combination averaged 18% that of drug-free cultures. The growth rate of GIC cells exposed to the drug combination increased to 30% of controls after Ara-C selection and to 50% after melphalan selection. Our results suggest that retroviral transfer of MFG/GIC may be useful for chemoprotection against the toxicities of nitrogen mustards and cytosine nucleoside analogs.

Gene amplification of human cytidine deaminase proviral cDNA and increased levels of its mRNA produces enhanced drug resistance to cytosine arabinoside in retroviral-transduced murine fibroblasts.

Hematopoietic toxicity is one of the major problems that limits the effectiveness of many antineoplastic drugs. One approach to overcome this problem is to confer chemoresistance to the hematopoietic cells by gene transfer of drug resistance genes. Human cytidine deaminase (CD) inactivates the cytosine nucleoside analogues, such as cytosine arabinoside (ARA-C), by deamination. We have reported previously that retroviral-mediated gene transfer of CD conferred drug resistance to ARA-C in murine cells. One of the major problems in the use of these vectors is to obtain adequate and prolonged expression of the transferred gene to produce a therapeutic effect in the transduced cells. The objective of this investigation was to determine if it is possible to increase the expression of CD proviral DNA in transduced murine fibroblast cells. We observed that by the use of continuous exposure to increasing concentrations of ARA-C it was possible to enhance drug resistance in the transduced cells. This drug resistance was found to be associated with increases in CD enzyme activity and CD proviral mRNA and by amplification of the proviral CD gene.

Coexpression of cytidine deaminase and mutant dihydrofolate reductase by a bicistronic retroviral vector confers resistance to cytosine arabinoside and methotrexate.

The transfer of a drug resistance gene into hematopoietic cells is an approach being investigated to overcome the problem of myelosuppression produced by anticancer drugs. Chemotherapeutic agents are often given in combination in order to increase their effectiveness. Consequently, there is an advantage in designing vectors for gene transfer that are capable of expressing two drug resistance genes. We have constructed a bicistronic retroviral vector, MFG-DHFR-IRES/CD, which contains the mutated human dihydrofolate reductase (DHFR) cDNA with a phenylalanine-to-serine substitution at codon 31 (F31S) and the human cytidine deaminase (CD) cDNA. Murine fibroblast and hematopoietic cells were transduced with this vector and evaluated for their resistance to methotrexate (MTX) and cytosine arabinoside (ARA-C). The transduced fibroblast cells showed high levels of resistance to MTX and to ARA-C as determined by a clonogenic assay. Using enzymatic assays, we observed a coordinate increase in resistance to MTX and DHFR enzyme activity following an ARA-C selection. In addition, MTX selection produced an increase in CD enzyme activity and ARA-C resistance. Murine hematopoietic cells transduced with the bicistronic vector also showed drug resistance to both MTX and ARA-C. Interestingly, the double-gene construct conferred an equivalent level of drug resistance compared with single-gene vectors bearing only CD or DHFR genes in the hematopoietic cells. These results demonstrate the potential of the MFG-DHFR-IRES/CD vector to confer drug resistance to both MTX and ARA-C and may have future application in chemoprotection of normal hematopoietic cells in patients with cancer.